RESUMO
Chloroplast-actin (cp-actin) filaments are crucial for light-induced chloroplast movement, and appear in the front region of moving chloroplasts when visualized using GFP-mouse Talin. They are short and thick, exist between a chloroplast and the plasma membrane, and move actively and rapidly compared to cytoplasmic long actin filaments that run through a cell. The average period during which a cp-actin filament was observed at the same position was less than 0.5 s. The average lengths of the cp-actin filaments calculated from those at the front region of the moving chloroplast and those around the chloroplast periphery after stopping the movement were almost the same, approximately 0.8 µm. Each cp-actin filament is shown as a dotted line consisting of 4-5 dots. The vector sum of cp-actin filaments in a moving chloroplast is parallel to the moving direction of the chloroplast, suggesting that the direction of chloroplast movement is regulated by the vector sum of cp-actin filaments. However, once the chloroplasts stopped moving, the vector sum of the cp-actin filaments around the chloroplast periphery was close to zero, indicating that the direction of movement was undecided. To determine the precise structure of cp-actin filaments under electron microscopy, Arabidopsis leaves and fern Adiantum capillus-veneris gametophytes were frozen using a high-pressure freezer, and observed under electron microscopy. However, no bundled microfilaments were found, suggesting that the cp-actin filaments were unstable even under high-pressure freezing.
Assuntos
Citoesqueleto de Actina , Arabidopsis , Cloroplastos , Luz , Cloroplastos/fisiologia , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Cloroplastos/ultraestrutura , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Adiantum/fisiologia , Adiantum/efeitos da radiação , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Actinas/metabolismo , MovimentoRESUMO
In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global-local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global-local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Citocinese , Nicotiana/ultraestrutura , Fuso Acromático/ultraestrutura , Citoesqueleto de Actina/fisiologia , Tamanho Celular , Células Cultivadas , Centrifugação , Proteínas de Fluorescência Verde , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fuso Acromático/fisiologia , Nicotiana/fisiologiaRESUMO
The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213 N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1.
Assuntos
Germinação/genética , Giberelinas/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Sequência de Bases , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Sementes/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
To study plant organs, it is necessary to investigate the three-dimensional (3D) structures of plants. In recent years, non-destructive measurements through computed tomography (CT) have been used to understand the 3D structures of plants. In this study, we use the Chrysanthemum seticuspe capitulum inflorescence as an example and focus on contact points between the receptacles and florets within the 3D capitulum inflorescence bud structure to investigate the 3D arrangement of the florets on the receptacle. To determine the 3D order of the contact points, we constructed slice images from the CT volume data and detected the receptacles and florets in the image. However, because each CT sample comprises hundreds of slice images to be processed and each C. seticuspe capitulum inflorescence comprises several florets, manually detecting the receptacles and florets is labor-intensive. Therefore, we propose an automatic contact point detection method based on CT slice images using image recognition techniques. The proposed method improves the accuracy of contact point detection using prior knowledge that contact points exist only around the receptacle. In addition, the integration of the detection results enables the estimation of the 3D position of the contact points. According to the experimental results, we confirmed that the proposed method can detect contacts on slice images with high accuracy and estimate their 3D positions through clustering. Additionally, the sample-independent experiments showed that the proposed method achieved the same detection accuracy as sample-dependent experiments.
RESUMO
Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state (13)C nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.
Assuntos
Celulase/genética , Celulase/metabolismo , Celulose/biossíntese , Celulose/química , Gluconacetobacter xylinus/metabolismo , Configuração de Carboidratos , Metabolismo dos Carboidratos , Gluconacetobacter xylinus/enzimologia , Gluconacetobacter xylinus/genética , Mutação , Ressonância Magnética Nuclear Biomolecular , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In vitro reconstitution of whole cellular events is one of the important goals in synthetic biology. Using a cell-free protein synthesis (CFPS) system reconstituted with human translation factors and chaperones, we reproduced the biogenesis of ß-actin, synthesis, folding, and polymerization in a test tube. This system enabled us to define which step of the ß-actin biogenesis was defective in genetic mutations related to diseases. Hence, the CFPS system reconstituted with human factors may be a useful tool for analyzing proteostasis in eukaryotes.
Assuntos
Actinas/genética , Chaperonas Moleculares/genética , Mutação/genética , Eucariotos/genética , Polimerização , Biossíntese de Proteínas/genética , Dobramento de Proteína , Proteostase/genética , Biologia Sintética/métodosRESUMO
X-ray micro-CT is one of the most useful techniques to examine 3D cellular architecture inside dry seeds. However, the examination of imbibed seeds is difficult because immersion in water causes a decline in the image quality. Here, we examined the use of ionic liquids for specimen preparation of chemically fixed imbibed seeds of Arabidopsis. We found that treatment with high concentrations of ionic liquids after osmium tetroxide fixation helped not only to prevent the structural damage caused by seed shrinkage, but also to preserve the image quality. Under these conditions, the cellular architecture of seeds was also well maintained.
Assuntos
Arabidopsis/ultraestrutura , Sementes/ultraestrutura , Microtomografia por Raio-X/métodos , Líquidos Iônicos/química , Tetróxido de Ósmio/químicaRESUMO
Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Microtúbulos/metabolismo , Proteínas de Plantas/metabolismo , Prófase , Anticorpos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dinitrobenzenos/farmacologia , Meristema/citologia , Microtúbulos/efeitos dos fármacos , Modelos Biológicos , Cebolas/citologia , Prófase/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Análise Espaço-Temporal , Sulfanilamidas/farmacologia , Tiazolidinas/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
Bacillus brevis (Brevibacillus parabrevis) ATCC 8185 synthesizes two kinds of antibiotic peptides, cyclopeptide tyrocidine and linear gramicidin. The production of linear gramicidin can be induced by the standard method (using a skim milk medium for pre-culture and beef broth for the main culture) employed for the induction of tyrocidine. In this study, we tried to determine the optimal growth medium for B. brevis ATCC 8185 for synthesizing linear gramicidin. The yield of linear gramicidin produced by the standard method was 3.11 microg/ml. When beef broth was used both as the pre-medium and the main medium, the yield of the antibiotic was only 0.59 microg/ml. To confirm the influence of skim milk, the strain was grown in a 1% skim milk medium. As a result, the amount of linear gramicidin produced reached 20.3 microg/ml. These findings show the importance of skim milk in the production of linear gramicidin. In the skim milk medium, the cells produced an extracellular protease 2 h before the linear gramicidin was expressed. The 1% skim milk medium pretreated by this protease did not allow the induction of linear gramicidin into the cells, and protease activity was not detected in the supernatant of the culture. When the cells were cultivated in a 1% egg albumin medium, protease activity from the supernatant of the culture was detected, but production of linear gramicidin was not observed. Therefore, a 1% casein medium was used for production of linear gramicidin. As a result, the yield of linear gramicidin produced in the medium reached 6.69 microg/ml. We concluded that a digested product of the extracellular protease from casein enhances linear gramicidin production.
Assuntos
Bacillus/efeitos dos fármacos , Bacillus/metabolismo , Caseínas/farmacologia , Gramicidina/biossíntese , Fragmentos de Peptídeos/farmacologia , Animais , Bacillus/química , Proteínas de Bactérias/metabolismo , Bovinos , Meios de Cultura/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Gramicidina/química , Peptídeo Hidrolases/metabolismo , Fatores de TempoRESUMO
Two xyloglucan-specific endo-beta-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley beta-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.
Assuntos
Glicosídeo Hidrolases/genética , Sequência de Aminoácidos , Bacillaceae/enzimologia , Bacillaceae/genética , Bacillaceae/crescimento & desenvolvimento , Bactérias/enzimologia , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismoRESUMO
A diastereomeric mixture and the four stereoisomers of 2,6-dimethyloctane-1,8-dioic acid (2), a copulation release pheromone of the cowpea weevil, Callosobruchus maculatus, were synthesized. The stereoisomeric purities of the four synthetic isomers of 2 were determined by the HPLC analyses of their bis-2-(2,3-anthracenedicarboximide)-1-cyclohexyl esters.
Assuntos
Ácidos Dicarboxílicos/síntese química , Atrativos Sexuais/síntese química , Gorgulhos/química , Monoterpenos Acíclicos , Cromatografia Líquida de Alta Pressão , Ácidos Dicarboxílicos/química , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Conformação Molecular , Oxirredução , Atrativos Sexuais/química , Espectrofotometria Infravermelho , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Acetan is a water-soluble polysaccharide produced by a bacterial cellulose (BC) producer, Acetobacter xylinum. An acetan-nonproducing mutant, EP1, was generated from wild-type A. xylinum BPR2001 by the disruption of aceA, which may act to catalyze the first step of the acetan biosynthetic pathway in this bacterium. EP1 produced less BC than the wild-type strain. However, when EP1 was cultured in a medium containing acetan, BC production was stimulated and the final yield of BC was equivalent to that of BPR2001. The culture broth containing acetan was more viscous and the free cell number was higher than that of the broth without the polysaccharide, so acetan may hinder the coagulation of BC in the broth. The addition of 1.5 g/l agar also increased BC production; we concluded that acetan and BC syntheses were not directly related on the genetic level.
Assuntos
Celulose/biossíntese , Gluconacetobacter xylinus/metabolismo , Polissacarídeos Bacterianos/metabolismo , Ágar/metabolismo , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Gluconacetobacter xylinus/genética , Glicosiltransferases/química , Glicosiltransferases/genética , Mutagênese , Reação em Cadeia da Polimerase , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/genética , ViscosidadeRESUMO
An ORF2 gene located upstream of the cellulose synthase (bcs) operon of Acetobacter xylinum BPR2001 was disrupted and a mutant (M2-2) was constructed. In static cultivation, the parent strain produced a tough, colorless, and insoluble cellulose pellicle, whereas M2-2 culture produced a thin, yellow, and fragile pellicle. The results of X-ray diffraction and 13C solid-state NMR indicated that the product of M2-2 is a mixture of cellulose I, cellulose II, and amorphous cellulose. The cellulose I to cellulose II ratio of the mixture was evaluated from the signal areas of C6 to be about 1:2. Electron microscopy revealed that the product of M2-2 included ribbon-like cellulose and irregularly shaped particles attached to the ribbons. On the other hand, the mutant complemented with plasmid pSA-ORF2/k containing the ORF2 gene and BPR2001 produced only cellulose I. These results indicate that the ORF2 gene is involved in the production and crystallization of cellulose I microfibrils by this microorganism.
Assuntos
Celulose/química , Genes Bacterianos , Acetobacter/genética , Configuração de Carboidratos , Celulose/ultraestrutura , Teste de Complementação Genética , Microscopia Eletrônica , Difração de Raios XRESUMO
Poplar calli transcribed two cellulase (endo-1,4-beta-glucanase) genes, PopCel1 and PopCel2, whose mRNAs were differentially located in the growing leaves of poplar during cell wall synthesis. Histochemical and RT-PCR analyses of promoter-GUS fusion gene activities in transgenic poplar demonstrated that PopCel1 promoter-derived GUS activity was localized in the petiole and leaf veins, whereas PopCel2 was confined to mesophyll cells and disappeared from the tip during the development of leaves. Autoradiography of the leaf showed that the radioactivity of [14C]sucrose incorporated into cellulose corresponded to the combination of the sucrose-induced tissue-specific patterns of PopCel1 and PopCel2. Interestingly, 2,6-dichlorobenzonitrile (DCB) not only inhibited the incorporation of the radioactivity into cellulose, but also repressed the induction of both cellulase genes. Suppression of cellulases by expression of PopCel1 antisense cDNA or co-suppression of PopCel1 mRNA by overexpression of PopCel1 sense cDNA reduced leaf growth. Therefore, we came to the conclusion that PopCel1 and PopCel2 probably function to promote leaf growth in poplar by the endohydrolysis of 1,4-beta-glucan.